Prolume Ltd

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Prolume Ltd

• Nanolight Technology Division
• POB 2746
• Pinetop, Arizona 85935 USA
• www.nanolight.com
• T-1-928-367-1200
• F-1-928-367-1205

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Properties of Coelenterazine Luciferases
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Renilla reniformis
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Renilla Luciferase
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Renilla Luciferase pH/NaCl
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Gaussia princeps a Copepod
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Gaussia princeps luciferase a new
coelenterazine-using luciferase
Prolume Bruce J. Bryan Christopher S.
Szent-Gyorgyi Gene G. Finley Byron Ballou
Carnegie Mellon University Gregory W.
Fisher Judy Montebellier
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Gaussia princeps luciferase
Isolated by expression cloning from a cDNA
library Molecular weight 19,900 (with signal
peptide) 17,900 (without) pH
optimum 7.8 11 cys in 185 residues
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Gaussia luciferase expression in CHO cells
Vectors Rluc pRL-CMV Gluc
pcDNA3/GL and /GL(CO)
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Gaussia luciferase expression in CHO cells
(expanded)
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Thus in CHO cells, Gaussia luciferase is 15-fold
more active than the commercially available
Renilla luciferase, and is 750-fold more active
after human codon optimization.
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Not retained in mammalian cells unless fused
with cell proteins (actively secreted?) Signal
peptide does not function in E. coli
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Useful Characteristics

• Resistant to pH extremes--survives exposure to pH
  3 or pH 11 overnight (0ºC).
• Thermal stability to 60ºC, 20 recovery after
  15 minutes at 99 ºC (0.5M NaCl, pH8).
• Active in presence of 1-5 nonionic detergents
  (NP-40, Triton X-100, Triton X-114, CHAPSO).
  Resists cholate, deoxycholate.
• Recovers activity after 7M Guanidine Chloride, 8M
  ureaNP-40.

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Enzyme Activity and Turnover The specific
activity of Guassia Luciferase calculated from
the initial (zero time) intensity in the presence
of a large excess of coelenterazine (10 ?M) was
1.24 x 1016 quanta/mg.s. Michaelis constant
was found to be about 1.3-1.5 ?M at zero time and
3.0 mM at 60 sec. The abnormal kinetic behavior
could indicate a product inhibition. If so,
buffer composition to minimize the inhibition
should be found.
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Gaussia activity after IEF in urea-NP-40
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SignalP-HMM prediction (euk models) Gaussia
luciferase
Cleavage prob.
1.0
n-region prob.
h-region prob.
c-region prob.
0.8
Prediction Signal peptide Signal peptide
probability 1.000 Signal anchor probability
0.000 Max cleavage site probability 0.980 at 18
0.6
Score
0.4
0.2
0.0
M
G
V
K
V
L
F
A
L
I
C
I
A
V
A
E
A
K
P
T
E
N
N
E
D
F
N
I
V
A
V
A
S
N
F
A
T
T
D
L
D
A
D
R
G
K
L
P
G
K
K
L
P
L
E
V
L
K
E
M
E
A
N
A
R
K
A
G
C
T
0
10
20
30
40
50
60
70
Position
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Effect of varying cation concentration on
reaction rate of Gaussia luciferase from E. coli
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